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Most DNA-based microbial source tracking (MST) approaches target host-associated organisms within the order Bacteroidales , but the gut microbiota of humans and other animals contain organisms from an array of other taxonomic groups that might provide indicators of fecal pollution sources. To discern between human and nonhuman fecal sources, we compared the V6 regions of the 16S rRNA genes detected in fecal samples from six animal hosts to those found in sewage (as a proxy for humans). We focused on 10 abundant genera and used oligotyping, which can detect subtle differences between rRNA gene sequences from ecologically distinct organisms. Our analysis showed clear patterns of differential oligotype distributions between sewage and animal samples. Over 100 oligotypes of human origin occurred preferentially in sewage samples, and 99 human oligotypes were sewage specific. Sequences represented by the sewage-specific oligotypes can be used individually for development of PCR-based assays or together with the oligotypes preferentially associated with sewage to implement a signature-based approach. Analysis of sewage from Spain and Brazil showed that the sewage-specific oligotypes identified in U.S. sewage have the potential to be used as global alternative indicators of human fecal pollution. Environmental samples with evidence of prior human fecal contamination had consistent ratios of sewage signature oligotypes that corresponded to the trends observed for sewage. Our methodology represents a promising approach to identifying new bacterial taxa for MST applications and further highlights the potential of the family Lachnospiraceae to provide human-specific markers. In addition to source tracking applications, the patterns of the fine-scale population structure within fecal taxa suggest a fundamental relationship between bacteria and their hosts.

Meiotic sex evolved early in the history of eukaryotes. Giardia duodenalis (syn. Giardia lamblia, Giardia intestinalis), a parasitic protist belonging to an early diverging lineage of eukaryotes, shows no cytological or physiological evidence of meiotic or sexual processes. Recent molecular analyses challenge the idea that G. duodenalis is a strictly clonal organism by providing evidence of recombination between homologous chromosomes within one subgroup (Assemblage A) of this species as well as genetic transfer from one subgroup to another (Assemblage A-B). Because recombination is not well documented and because it is not known whether the observed inter-assemblage transfer represents true reciprocal genetic exchange or a non-sexual process, we analyzed genic sequences from all major subgroups (Assemblages A-G) of this species. For all assemblages, we detected molecular signatures consistent with meiotic sex or genetic exchange, including low levels of heterozygosity, as indicated by allelic sequence divergence within isolates, and intra- and inter-assemblage recombination. The identification of recombination between assemblages suggests a shared gene pool and calls into question whether it is appropriate to divide the genetically distinct assemblages of G. duodenalis into a species complex.

The human distal gut harbours a vast ensemble of microbes (the microbiota) that provide important metabolic capabilities, including the ability to extract energy from otherwise indigestible dietary polysaccharides. Studies of a few unrelated, healthy adults have revealed substantial diversity in their gut communities, as measured by sequencing 16S rRNA genes, yet how this diversity relates to function and to the rest of the genes in the collective genomes of the microbiota (the gut microbiome) remains obscure. Studies of lean and obese mice suggest that the gut microbiota affects energy balance by influencing the efficiency of calorie harvest from the diet, and how this harvested energy is used and stored. Here we characterize the faecal microbial communities of adult female monozygotic and dizygotic twin pairs concordant for leanness or obesity, and their mothers, to address how host genotype, environmental exposure and host adiposity influence the gut microbiome. Analysis of 154 individuals yielded 9,920 near full-length and 1,937,461 partial bacterial 16S rRNA sequences, plus 2.14 gigabases from their microbiomes. The results reveal that the human gut microbiome is shared among family members, but that each person's gut microbial community varies in the specific bacterial lineages present, with a comparable degree of co-variation between adult monozygotic and dizygotic twin pairs. However, there was a wide array of shared microbial genes among sampled individuals, comprising an extensive, identifiable 'core microbiome' at the gene, rather than at the organismal lineage, level. Obesity is associated with phylum-level changes in the microbiota, reduced bacterial diversity and altered representation of bacterial genes and metabolic pathways. These results demonstrate that a diversity of organismal assemblages can nonetheless yield a core microbiome at a functional level, and that deviations from this core are associated with different physiological states (obese compared with lean). ©2009 Macmillan Publishers Limited. All rights reserved.

The genome of the eukaryotic protist Giardia lamblia, an important human intestinal parasite, is compact in structure and content, contains few introns or mitochondrial relics, and has simplified machinery for DNA replication, transcription, RNA processing, and most metabolic pathways. Protein kinases comprise the single largest protein class and reflect Giardia's requirement for a complex signal transduction network for coordinating differentiation. Lateral gene transfer from bacterial and archaeal donors has shaped Giardia's genome, and previously unknown gene families, for example, cysteine-rich structural proteins, have been discovered. Unexpectedly, the genome shows little evidence of heterozygosity, supporting recent speculations that this organism is sexual. This genome sequence will not only be valuable for investigating the evolution of eukaryotes, but will also be applied to the search for new therapeutics for this parasite.

The Deepwater Horizon (DWH) oil well blowout generated an enormous plume of dispersed hydrocarbons that substantially altered the Gulf of Mexico’s deep-sea microbial community. A significant enrichment of distinct microbial populations was observed, yet, little is known about the abundance and richness of specific microbial ecotypes involved in gas, oil and dispersant biodegradation in the wake of oil spills. Here, we document a previously unrecognized diversity of closely related taxa affiliating with Cycloclasticus, Colwellia and Oceanospirillaceae and describe their spatio-temporal distribution in the Gulf’s deepwater, in close proximity to the discharge site and at increasing distance from it, before, during and after the discharge. A highly sensitive, computational method (oligotyping) applied to a data set generated from 454-tag pyrosequencing of bacterial 16S ribosomal RNA gene V4–V6 regions, enabled the detection of population dynamics at the sub-operational taxonomic unit level (0.2% sequence similarity). The biogeochemical signature of the deep-sea samples was assessed via total cell counts, concentrations of short-chain alkanes (C1–C5), nutrients, (colored) dissolved organic and inorganic carbon, as well as methane oxidation rates. Statistical analysis elucidated environmental factors that shaped ecologically relevant dynamics of oligotypes, which likely represent distinct ecotypes. Major hydrocarbon degraders, adapted to the slow-diffusive natural hydrocarbon seepage in the Gulf of Mexico, appeared unable to cope with the conditions encountered during the DWH spill or were outcompeted. In contrast, diverse, rare taxa increased rapidly in abundance, underscoring the importance of specialized sub-populations and potential ecotypes during massive deep-sea oil discharges and perhaps other large-scale perturbations.

The coalescence of next-generation DNA sequencing methods, ecological perspectives, and bioinformatics analysis tools is rapidly advancing our understanding of the evolution and function of vertebrate-associated bacterial communities. Delineation of host-microbe associations has applied benefits ranging from clinical treatments to protecting our natural waters. Microbial communities follow some broad-scale patterns observed for macroorganisms, but it remains unclear how the specialization of intestinal vertebrate-associated communities to a particular host environment influences broad-scale patterns in microbial abundance and distribution. We analyzed the V6 region of 16S rRNA genes amplified from 106 fecal samples spanning Aves , Mammalia , and Actinopterygii (ray-finned fish). We investigated the interspecific abundance-occupancy relationship, where widespread taxa tend to be more abundant than narrowly distributed taxa, among operational taxonomic units (OTUs) within and among host species. In a separate analysis, we identified specialist OTUs that were highly abundant in a single host and rare in all other hosts by using a multinomial model without excluding undersampled OTUs a priori . We show that intestinal microbes in humans and other vertebrates display abundance-occupancy relationships, but because intestinal host-associated communities have undergone intense specialization, this trend is violated by a disproportionately large number of specialist taxa. Although it is difficult to distinguish the effects of dispersal limitations, host selection, historical contingency, and stochastic processes on community assembly, results suggest that intestinal bacteria can be shared among diverse hosts in ways that resemble the distribution of “free-living” bacteria in the extraintestinal environment.

Molecular characterizations of the gut microbiome from individual human stool samples have identified community patterns that correlate with age, disease, diet, and other human characteristics, but resources for marker gene studies that consider microbiome trends among human populations scale with the number of individuals sampled from each population. As an alternative strategy for sampling populations, we examined whether sewage accurately reflects the microbial community of a mixture of stool samples. We used oligotyping of high-throughput 16S rRNA gene sequence data to compare the bacterial distribution in a stool data set to a sewage influent data set from 71 U.S. cities. On average, only 15% of sewage sample sequence reads were attributed to human fecal origin, but sewage recaptured most (97%) human fecal oligotypes. The most common oligotypes in stool matched the most common and abundant in sewage. After informatically separating sequences of human fecal origin, sewage samples exhibited ~3× greater diversity than stool samples. Comparisons among municipal sewage communities revealed the ubiquitous and abundant occurrence of 27 human fecal oligotypes, representing an apparent core set of organisms in U.S. populations. The fecal community variability among U.S. populations was significantly lower than among individuals. It clustered into three primary community structures distinguished by oligotypes from either: Bacteroidaceae , Prevotellaceae , or Lachnospiraceae/Ruminococcaceae . These distribution patterns reflected human population variation and predicted whether samples represented lean or obese populations with 81 to 89% accuracy. Our findings demonstrate that sewage represents the fecal microbial community of human populations and captures population-level traits of the human microbiome. IMPORTANCE The gut microbiota serves important functions in healthy humans. Numerous projects aim to define a healthy gut microbiome and its association with health states. However, financial considerations and privacy concerns limit the number of individuals who can be screened. By analyzing sewage from 71 cities, we demonstrate that geographically distributed U.S. populations share a small set of bacteria whose members represent various common community states within U.S. adults. Cities were differentiated by their sewage bacterial communities, and the community structures were good predictors of a city's estimated level of obesity. Our approach demonstrates the use of sewage as a means to sample the fecal microbiota from millions of people and its potential to elucidate microbiome patterns associated with human demographics.

Summary Sulfate reduction, mediated by sulfate-reducing bacteria (SRB), is the dominant remineralization pathway in sediments of New England salt marshes. High sulfate reduction rates are associated with the rhizosphere of Spartina alterniflora when plants elongate aboveground. The growth process concurrently produces significant amounts of new rhizome material belowground and the plants leak dissolved organic compounds. This study investigated the diversity of SRB in a salt marsh over an annual growth cycle of S. alterniflora by exploring the diversity of a functional gene, dissimilatory sulfite reductase (dsrAB). Because the dsrAB gene is a key gene in the anaerobic sulfate-respiration pathway, it allows the identification of microorganisms responsible for sulfate reduction. Conserved dsrAB primers in polymerase chain reaction (PCR) generated full-length dsrAB amplicons for cloning and DNA sequence analysis. Nearly 80% of 380 clone sequences were similar to genes from Desulfosarcina and Desulfobacterium species within Desulfobacteraceae. This reinforces the hypothesis that complete oxidizers with high substrate versatility dominate the marsh. However, the phylotypes formed several clades that were distinct from cultured representatives, indicating a greater diversity of SRB than previously appreciated. Several dsrAB sequences were related to homologues from Gram-positive, thermophilic and non-thermophilic Desulfotomaculum species. One dsrAB lineage formed a sister group to cultured members of the delta-proteobacterial group Syntrophobacteraceae. A deeply branching dsrAB lineage was not affiliated with genes from any cultured SRB. The sequence data from this study will allow for the design of probes or primers that can quantitatively assess the diverse range of sulfate reducers present in the environment.

The methanogenic community in hydrothermally active sediments of Guaymas Basin (Gulf of California, Mexico) was analyzed by PCR amplification, cloning, and sequencing of methyl coenzyme M reductase ( mcrA ) and 16S rRNA genes. Members of the Methanomicrobiales and Methanosarcinales dominated the mcrA and 16S rRNA clone libraries from the upper 15 cm of the sediments. Within the H 2 /CO 2 - and formate-utilizing family Methanomicrobiales , two mcrA and 16S rRNA lineages were closely affiliated with cultured species of the genera Methanoculleus and Methanocorpusculum . The most frequently recovered mcrA PCR amplicons within the Methanomicrobiales did not branch with any cultured genera. Within the nutritionally versatile family Methanosarcinales , one 16S rRNA amplicon and most of the mcrA PCR amplicons were affiliated with the obligately acetate utilizing species Methanosaeta concilii . The mcrA clone libraries also included phylotypes related to the methyl-disproportionating genus Methanococcoides . However, two mcrA and two 16S rRNA lineages within the Methanosarcinales were unrelated to any cultured genus. Overall, the clone libraries indicate a diversified methanogen community that uses H 2 /CO 2 , formate, acetate, and methylated substrates. Phylogenetic affiliations of mcrA and 16S rRNA clones with thermophilic and nonthermophilic cultured isolates indicate a mixed mesophilic and thermophilic methanogen community in the surficial Guaymas sediments.

Through a genome survey and phylogenetic analysis, we have identified and sequenced 14 new coding regions for alpha-giardins in Giardia lamblia. These proteins are related to annexins and comprise a multi-gene family with 21 members. Many alpha giardins are highly expressed proteins that are very immunogenic during acute giardiasis in humans. However, little is known about the function of these proteins. By using PCR with different combinations of gene-specific primers, we demonstrated that several of the genes localised to the same chromosomal fragment. These data point towards a molecular evolution through gene duplication and subsequent functional divergence. Semi-quantitative reverse transcriptase-PCR analysis of the Giardia life cycle revealed large differences in mRNA expression levels of the alpha giardins. Epitope tagging of the alpha-giardins localised them to different cytoskeletal components, such as the flagella and the adhesive disc, but also to the plasma membrane. These localisation experiments suggest alpha-giardins play a role in cell motility, attachment and membrane stability.

Enzymes in the cytochrome P450 gene family 1 (CYP1) catalyze the metabolic activation of numerous hydrocarbon carcinogens and various natural compounds. CYP1 family members have been identified in several vertebrates, including fish, amphibians, birds, and mammals, and are inducible by aromatic hydrocarbons acting through the aryl hydrocarbon receptor (AHR). Together with its heterodimeric partner ARNT, the ligand-bound AHR binds conserved xenobiotic response elements (XREs) near the promoter of CYP1A and other genes. However, some populations of the Atlantic killifish Fundulus heteroclitus inhabiting highly contaminated sites are refractory to CYP1A induction by aromatic hydrocarbons. To better understand the mechanisms underlying this phenomenon, we are characterizing the AHR-CYP1A signaling pathway in this species. We report here the characterization of a genomic clone containing the 5(') end of the wild-type F. heteroclitus CYP1A gene. The 5(') coding sequence matches that of the F. heteroclitus CYP1A cDNA reported earlier [Comp. Biochem. Physiol. 121C (1998) 231]. Consistent with its inducibility by AHR agonists, the CYP1A gene contains three consensus XREs (5(')CACGC3(')) within 1.6 kb of the putative transcriptional start site. When oligonucleotides containing each of these sites were analyzed in an electrophoretic mobility shift assay, one of these showed a strong, TCDD-inducible mobility shift in the presence of in vitro expressed mouse AHR protein. These sequence data and initial functional characterization provide a valuable tool for the study of genetic variations in CYP1A expression and activity in sensitive and resistant populations. These studies may ultimately shed light on the importance of P4501A activity in xenobiotic toxicity.

In conclusion, the present study confirms that genetic diversity exists among Blastocystis organisms isolated from different hosts and suggests that more than one species of Blastocystis infect humans. The possibility of cross-contamination between animals and humans exists, and the data suggest a low host specificity of these microorganisms. Thus, if Blastocystis isolated from animals are capable of infecting humans, the number of animals found to be infected with Blastocystis could represent a large potential reservoir for infection of humans.

Functional assays of genes have historically led to insights about the activities of a protein or protein cascade. However, the rapid expansion of genomic and proteomic information for a variety of diverse taxa is an alternative and powerful means of predicting function by comparing the enzymes and metabolic pathways used by different organisms. As part of the Giardia lamblia genome sequencing project, we routinely survey the complement of predicted proteins and compare those found in this putatively early diverging eukaryote with those of prokaryotes and more recently evolved eukaryotic lineages. Such comparisons reveal the minimal composition of conserved metabolic pathways, suggest which proteins may have been acquired by lateral transfer, and, by their absence, hint at functions lost in the transition from a free-living to a parasitic lifestyle. Here, we describe the use of bioinformatic approaches to investigate the complement and conservation of proteins in Giardia involved in the regulation of translation. We compare an FK506 binding protein homologue and phosphatidylinositol kinase-related kinase present in Giardia to those found in other eukaryotes for which complete genomic sequence data are available. Our investigation of the Giardia genome suggests that PIK-related kinases are of ancient origin and are highly conserved.

To investigate the complexity of the endomembrane transport system in the early diverging eukaryote, Giardia lamblia , we characterized homologues of the GTP-binding proteins, Rab1 and Rab2, involved in regulating vesicular trafficking between the endoplasmic reticulum and Golgi in higher eukaryotes, and GDI, which plays a key role in the cycling of Rab proteins. G. lamblia Rab1, 2.1, and GDI sequences largely resemble yeast and mammalian homologues, are transcribed as 0.66-, 0.62-, and 1.4-kb messages, respectively, and are expressed during growth and encystation. Western analyses detected an abundant Rab/GDI complex at approximately 80 kDa, and free GDI (60 kDa) in both trophozoites and encysting cells. Immunoelectron microscopy with antibody to Rab1 localized Rab with ER, encystation secretory vesicles, and lysosome-like peripheral vesicles. GDI associated with these structures, and with small vesicles found throughout the cytoplasm, consistent with GDI’s key role in Rab cycling between organelles within the cell. Index Descriptives and Abbreviations : Giardia , Diplomonadida; GDI, guanine dissociation inhibitor; GEF, guanine nucleotide exchange factor; GAP, GTPase activating protein; ER, endoplasmic reticulum; ORF, opening reading frame; PCR, polymerase chain reaction; SSC, sodium, sodium citrate; ECL, enhanced chemiluminescence; PBS, phosphate buffered saline; CHAPS, 3-[3-(cholamidopropyl)dimethylammonio]-1propanesulfonate; SDS–PAGE, sodium dodecyl sulfate–polyacrylamide gel electrophoresis; DNA, deoxyribonucleic acid; Tyr, tyrosine; Gln, glutamine; Arg, arginine; Thr, threonine; Met, methionine; ESV, encystation secretory vesicles; PV, peripheral vesicles.

Giardia lamblia and Entamoeba histolytica are amitochondriate, microaerophilic protists which use fermentation enzymes like those of bacteria to survive anaerobic conditions within the intestinal lumen. Genes encoding fermentation enzymes and related electron transport peptides (e.g., ferredoxins) in giardia organisms and amebae are hypothesized to be derived from either an ancient anaerobic eukaryote (amitochondriate fossil hypothesis), a mitochondrial endosymbiont (hydrogen hypothesis), or anaerobic bacteria (lateral transfer hypothesis). The goals here were to complete the molecular characterization of giardial and amebic fermentation enzymes and to determine the origins of the genes encoding them, when possible. A putative giardia [2Fe-2S]ferredoxin which had a hypothetical organelle-targeting sequence at its N terminus showed similarity to mitochondrial ferredoxins and the hydrogenosomal ferredoxin of Trichomonas vaginalis (another luminal protist). However, phylogenetic trees were star shaped, with weak bootstrap support, so we were unable to confirm or rule out the endosymbiotic origin of the giardia [2Fe-2S]ferredoxin gene. Putative giardial and amebic 6-kDa ferredoxins, ferredoxin-nitroreductase fusion proteins, and oxygen-insensitive nitroreductases each tentatively supported the lateral transfer hypothesis. Although there were not enough sequences to perform meaningful phylogenetic analyses, the unique common occurrence of these peptides and enzymes in giardia organisms, amebae, and the few anaerobic prokaryotes suggests the possibility of lateral transfer. In contrast, there was more robust phylogenetic evidence for the lateral transfer of G . lamblia genes encoding an NADH oxidase from a gram-positive coccus and a microbial group 3 alcohol dehydrogenase from thermoanaerobic prokaryotes. In further support of lateral transfer, the G . lamblia NADH oxidase and adh3 genes appeared to have an evolutionary history distinct from those of E . histolytica .

Microbial communities in hydrothermally active sediments of the Guaymas Basin (Gulf of California, Mexico) were studied by using 16S rRNA sequencing and carbon isotopic analysis of archaeal and bacterial lipids. The Guaymas sediments harbored uncultured euryarchaeota of two distinct phylogenetic lineages within the anaerobic methane oxidation 1 (ANME-1) group, ANME-1a and ANME-1b, and of the ANME-2c lineage within the Methanosarcinales , both previously assigned to the methanotrophic archaea. The archaeal lipids in the Guaymas Basin sediments included archaeol, diagnostic for nonthermophilic euryarchaeota, and sn -2-hydroxyarchaeol, with the latter compound being particularly abundant in cultured members of the Methanosarcinales . The concentrations of these compounds were among the highest observed so far in studies of methane seep environments. The δ- 13 C values of these lipids (δ- 13 C = −89 to −58‰) indicate an origin from anaerobic methanotrophic archaea. This molecular-isotopic signature was found not only in samples that yielded predominantly ANME-2 clones but also in samples that yielded exclusively ANME-1 clones. ANME-1 archaea therefore remain strong candidates for mediation of the anaerobic oxidation of methane. Based on 16S rRNA data, the Guaymas sediments harbor phylogenetically diverse bacterial populations, which show considerable overlap with bacterial populations of geothermal habitats and natural or anthropogenic hydrocarbon-rich sites. Consistent with earlier observations, our combined evidence from bacterial phylogeny and molecular-isotopic data indicates an important role of some novel deeply branching bacteria in anaerobic methanotrophy. Anaerobic methane oxidation likely represents a significant and widely occurring process in the trophic ecology of methane-rich hydrothermal vents. This study stresses a high diversity among communities capable of anaerobic oxidation of methane.

We studied the evolutionary relationships among basal metazoan lineages by using complete large subunit (LSU) and small subunit (SSU) ribosomal RNA sequences for 23 taxa. After identifying competing hypotheses, we performed maximum likelihood searches for trees conforming to each hypothesis. Kishino–Hasegawa tests were used to determine whether the data (LSU, SSU, and combined) reject any of the competing hypotheses. We also conducted unconstrained tree searches, compared the resulting topologies, and calculated bootstrap indices. Shimodaira–Hasegawa tests were applied to determine whether the data reject any of the topologies resulting from the constrained and unconstrained tree searches. LSU, SSU, and the combined data strongly contradict two assertions pertaining to sponge phylogeny. Hexactinellid sponges are not likely to be the basal lineage of a monophyletic Porifera or the sister group to all other animals. Instead, Hexactinellida and Demospongia form a well-supported clade of siliceous sponges, Silicea. It remains unclear, on the basis of these data alone, whether the calcarean sponges are more closely related to Silicea or to nonsponge animals. The SSU and combined data reject the hypothesis that Bilateria is more closely related to Ctenophora than it is to Cnidaria, whereas LSU data alone do not refute either hypothesis. LSU and SSU data agree in supporting the monophyly of Bilateria, Cnidaria, Ctenophora, and Metazoa. LSU sequence data reveal phylogenetic structure in a data set with limited taxon sampling. Continued accumulation of LSU sequences should increase our understanding of animal phylogeny.

Jakobids are free-living, heterotrophic flagellates that might represent early-diverging mitochondrial protists. They share ultrastructural similarities with eukaryotes that occupy basal positions in molecular phylogenies, and their mitochondrial genome architecture is eubacterial-like, suggesting a close affinity with the ancestral alpha-proteobacterial symbiont that gave rise to mitochondria and hydrogenosomes. To elucidate relationships among jakobids and other early-diverging eukaryotic lineages, we characterized alpha- and beta-tubulin genes from four jakobids: Jakoba libera, Jakoba incarcerata, Reclinomonas americana (the "core jakobids"), and Malawimonas jakobiformis. These are the first reports of nuclear genes from these organisms. Phylogenies based on alpha-, beta-, and combined alpha- plus beta-tubulin protein data sets do not support the monophyly of the jakobids. While beta-tubulin and combined alpha- plus beta-tubulin phylogenies showed a sister group relationship between J. libera and R. americana, the two other jakobids, M. jakobiformis and J. incarcerata, had unclear affinities. In all three analyses, J. libera, R. americana, and M. jakobiformis emerged from within a well-supported large "plant-protist" clade that included plants, green algae, cryptophytes, stramenopiles, alveolates, Euglenozoa, Heterolobosea, and several other protist groups, but not animals, fungi, microsporidia, parabasalids, or diplomonads. A preferred branching order within the plant-protist clade was not identified, but there was a tendency for the J. libera-R. americana lineage to group with a clade made up of the heteroloboseid amoeboflagellates and euglenozoan protists. Jakoba incarcerata branched within the plant-protist clade in the beta- and the combined alpha- plus beta-tubulin phylogenies. In alpha- tubulin trees, J. incarcerata occupied an unresolved position, weakly grouping with the animal/fungal/microsporidian group or with amitochondriate parabasalid and diplomonad lineages, depending on the phylogenetic method employed. Tubulin gene phylogenies were in general agreement with mitochondrial gene phylogenies and ultrastructural data in indicating that the "jakobids" may be polyphyletic. Relationships with the putatively deep-branching amitochondriate diplomonads remain uncertain.

Sexually mature female squid Loligo pealei harbour dense bacterial communities in their accessory nidamental glands (ANGs) and in their egg capsules (ECs). This study describes a molecular approach using the 16S rRNA gene (rDNA) to identify bacterial populations within the ANG and the ECs of the North Atlantic squid species L. pealei. Fluorescent in situ hybridization (FISH) and 16S rDNA analysis showed that predominantly alpha- and, to a lesser extent, gamma-proteobacteria were the predominant components of the ANG and EC bacterial communities. Sequencing results showed the presence of alpha-proteobacterial populations affiliated with the Roseobacter group and additional deep-branching alpha-proteobacterial lineages. In contrast, isolates from the ANG and ECs contained only a few alpha-proteobacteria of the Roseobacter group compared with several gamma-proteobacterial isolates, mostly Shewanella and Pseudoalteromonas species. Most of the ANG-associated bacterial populations were also found within the ECs of L. pealei. The molecular approach allowed the visualization of alpha-proteobacteria as major constituents of a bacterial symbiosis within the reproductive system of the Loliginidae.

Leptomyxid amoebae encompass a diverse assemblage of amoeboid protists that have been implicated as encephalitis-causing agents. This characteristic is attributed to recent studies identifying new members of the Leptomyxidae, in particular, Balamuthia mandrillaris, that cause the disease. Their morphologies range from limax to plasmodial, as well as reticulated and polyaxial. Although systematic studies have identified B. mandrillaris as a new member of the Leptomyxidae, its precise placement within the leptomyxids is uncertain. To further assess the taxonomic placement of Balamuthia among the leptomyxid amoebae and to determine whether the members of the Leptomyxida form a monophyletic assemblage, we have sequenced 16S-like rRNA genes from representatives of three leptomyxid families. Our phylogenetic analyses revealed that current members of the order Leptomyxida do not constitute a monophyletic assemblage. Our analyses clearly show that Gephyramoeba, as well as Balamuthia do not belong in the order Leptomyxida. We highlight where molecular data give differing insights than taxonomic schemes based on traditional characters.

Strict one-to-one correspondence between codons and amino acids is thought to be an essential feature of the genetic code. However, we report that one codon can code for two different amino acids with the choice of the inserted amino acid determined by a specific 3′ untranslated region structure and location of the dual-function codon within the messenger RNA (mRNA). We found that the codon UGA specifies insertion of selenocysteine and cysteine in the ciliate Euplotes crassus , that the dual use of this codon can occur even within the same gene, and that the structural arrangements of Euplotes mRNA preserve location-dependent dual function of UGA when expressed in mammalian cells. Thus, the genetic code supports the use of one codon to code for multiple amino acids.

Since we published a phylogenetic analysis of the CYP1A subfamily in 1995, several additional full-length sequences have been reported, including three members of an entirely new subfamily, CYP1B. Two avian sequences were recently published, so that CYP1A sequence data are now available from three of the five major vertebrate lineages. The two new branches that have been added to the CYP1 family tree significantly add to our understanding of P450 evolution. The inclusion of the CYP1Bs to the phylogenetic analysis allows us to root inferred trees. Addition of the avian CYP1As indicates that the CYP1A1/CYP1A2 duplication present in the mammalian lineage may have occurred after the divergence of birds and mammals. The number of fish species from which full-length coding regions of CYP1A genes have been sequenced has increased from four (trout, plaice, toadfish, and scup) to nine. These include CYP1A sequences from tomcod, butterflyfish, sea bream, sea bass, and the full-length sequence of CYP1A from the killifish Fundulus heteroclitus that is reported here. Phylogenetic analyses incorporating the new fish CYP1A sequences support our original conclusion that the fish CYP1As are monophyletic and indicate that the genes are evolving at very different rates in different species.

Polycystine radiolaria are among few protistan groups that possess a comprehensive fossil record available for study by micropaleontologists. The Polycystinea and the Acantharea, whose skeletons do not become fossilized, were once members of the class “Radiolaria” (“Radiolaria” sensu lato : Polycystinea, Phaeodarea, and Acantharea) originally proposed by Haeckel but are now included in the superclass Actinopoda. Phylogenetic relationships within this superclass remain largely enigmatic. We investigated the evolutionary relationship of the Acantharea and the Polycystinea to other protists using phylogenetic analyses of 16S-like ribosomal RNA (rRNA) coding regions. We circumvented the need to culture these organisms by collecting and maintaining reproductive stages that contain many copies of their genomic DNA. This strategy facilitated extraction of genomic DNA and its purification from symbiont and prey DNA. Phylogenetic trees inferred from comparisons of 16S-like coding regions do not support a shared history between the Acantharea and the Polycystinea. However, the monophyly of the Acantharea and the separate monophyly of the Polycystinea (Spumellarida) are well supported by our molecular-based trees. The acantharian lineage branches among crown organisms whereas the polycystine lineage diverges before the radiation of the crown groups. We conclude that the Actinopoda does not represent a monophyletic evolutionary assemblage and recommend that this taxonomic designation be discarded.

The small subunit rRNA gene sequences of the karyorelictean ciliates, Loxodes striatus and Protocruzia sp., and the heterotrichian ciliates, Climacostomum virens and Eufolliculina uhligi, were used to test the evolution of nuclear dualism in the Phylum Ciliophora. Phylogenies derived using a least squares distance method, neighbour joining, and maximum parsimony demonstrate that the karyorelictean ciliates sensu Small and Lynn, 1985 do not form a monophyletic group. However, Loxodes and the heterotrich ciliates form the first branch in the ciliate lineage, and Protocruzia branches, in distance methods, basal to the spirotrich lineage. It is proposed that Protocruzia be removed from the Class Karyorelictea, and placed in closer taxonomic association with the spirotrich lineage. The distribution of nuclear division types along the phylogenetic tree is consistent with the notion that macronuclei incapable of division represent a derived rather than a primitive or "karyorelictid" character trait.

Faecal pollution contains a rich and diverse community of bacteria derived from animals and humans, many of which might serve as alternatives to the traditional enterococci and Escherichia coli faecal indicators. We used massively parallel sequencing (MPS) of the 16S rRNA gene to characterize microbial communities from wastewater treatment plant (WWTP) influent sewage from 12 cities geographically distributed across the USA. We examined members of the Clostridiales, which included the families Clostridiaceae, Lachnospiraceae and Ruminococcaceae for their potential as sewage indicators. Lachnospiraceae was one of the most abundant groups of faecal bacteria in sewage, and several Lachnospiraceae high-abundance sewage pyrotags occurred in at least 46 of 48 human faecal samples. Clone libraries targeting Clostridium coccoides (C. coccoides) in sewage samples demonstrated that Lachnospiraceae-annotated V6 pyrotags encompassed the previously reported C. coccoides group. We used oligotyping to profile the genus Blautia within Lachnospiraceae and found oligotypes comprised of 24 entropy components that showed patterns of host specificity. These findings suggest that indicators based on Blautia might have the capacity to discriminate between different faecal pollution sources. Development of source-specific alternative indicators would enhance water quality assessments, which leads to improved ecosystem health and reduced human health risk due to waterborne disease.

Background Neonatal sepsis due to intestinal bacterial translocation is a major cause of morbidity and mortality. Understanding microbial colonisation of the gut in prematurity may predict risk of sepsis to guide future strategies to manipulate the microbiome. Methods Prospective longitudinal study of premature infants. Stool samples were obtained weekly. DNA was extracted and the V6 hypervariable region of 16S rRNA was amplified followed by high throughput pyrosequencing, comparing subjects with and without sepsis. Results Six neonates were 24–27 weeks gestation at birth and had 18 samples analysed. Two subjects had no sepsis during the study period, two developed late-onset culture-positive sepsis and two had culture-negative systemic inflammation. 324 350 sequences were obtained. The meconium was not sterile and had predominance of Lactobacillus, Staphylococcus and Enterobacteriales. Overall, infants who developed sepsis began life with low microbial diversity, and acquired a predominance of Staphylococcus , while healthy infants had more diversity and predominance of Clostridium, Klebsiella and Veillonella . Conclusions In very low birth weight infants, the authors found that meconium is not sterile and is less diverse from birth in infants who will develop late-onset sepsis. Empiric, prolonged antibiotics profoundly decrease microbial diversity and promote a microbiota that is associated not only with neonatal sepsis, but the predominant pathogen previously identified in the microbiome. Our data suggest that there may be a ‘healthy microbiome’ present in extremely premature neonates that may ameliorate risk of sepsis. More research is needed to determine whether altered antibiotics, probiotics or other novel therapies can re-establish a healthy microbiome in neonates.

The small subunit ribosomal RNA (rRNA) of the anaerobic amoeba Phreatamoeba balamuthi is the longest 16S-like rRNA sequenced to date. Secondary structure analysis suggests that the additional sequence is incorporated in canonical eukaryotic expansion regions and is not due to the presence of introns. Reverse transcriptase sequencing of total RNA extracts confirmed that two uncommonly long expansion regions are present in native P. balamuthi 16S-like rRNA. Primary sequence comparison and similar secondary structure indicate a 61 base stem and loop repeat within an expansion region; a mechanism whereby the repeat may have been incorporated is presented. P. balamuthi provides further evidence that 16S-like rRNA length does not correlate with phylogenetic position.

The amoebae, a phenotypically diverse, paraphyletic group of protists, have been largely neglected by molecular phylogeneticists. To better understand the evolution of amoebae, we sequenced and analyzed the 16S-like ribosomal RNA genes of three vahlkampfiid amoebae: Paratetramitus jugosus, Tetramitus rostratus and Vahlkampfia lobospinosa. The Vahlkampfiidae lineage is monophyletic, branches early along the eukaryotic line of descent, and is not a close relative of the multicellular amoebae that also reversibly transform from amoebae to flagellates.

Nucleotide sequences of the small subunit ribosomal RNA (18S) gene were used to investigate evolutionary relationships within the Fungi. The inferred tree topologies are in general agreement with traditional classifications in the following ways: (1) the Chytridiomycota and Zygomycota appear to be basal groups within the Fungi. (2) The Ascomycota and Basidiomycota are a derived monophyletic group. (3) Relationships within the Ascomycota are concordant with traditional orders and divide the hemi- and euascomycetes into distinct lineages. (4) The Basidiomycota is divided between the holobasidiomycetes and phragmobasidiomycetes. Conflicts with traditional classification were limited to weakly supported branches of the tree. Strongly supported relationships were robust to minor changes in alignment, method of analysis, and various weighting schemes. Weighting, either of transversions or by site, did not convincingly improve the status of poorly supported portions of the tree. The rate of variation at particular sites does not appear to be independent of lineage, suggesting that covariation of sites may be an important phenomenon in these genes.

Owing to the extreme salinity ( approximately 10 times saltier than the oceans), near toxic magnesium levels (approximately 2.0 M Mg(2+)), the dominance of divalent cations, acidic pH (6.0) and high-absorbed radiation flux rates, the Dead Sea represents a unique and harsh ecosystem. Measures of microbial presence (microscopy, pigments and lipids) indicate that during rare bloom events after exceptionally rainy seasons, the microbial communities can reach high densities. However, most of the time, when the Dead Sea level is declining and halite is precipitating from the water column, it is difficult to reliably measure the presence of microorganisms and their activities. Although a number of halophilic Archaea have been previously isolated from the Dead Sea, polar lipid analyses of biomass collected during Dead Sea blooms suggested that these isolates were not the major components of the microbial community of these blooms. In this study, in an effort to characterize the perennial microbial community of the Dead Sea and compare it with bloom assemblages, we performed metagenomic analyses of concentrated biomass from hundreds of liters of brine and of microbial material from the last massive Dead Sea bloom. The difference between the two conditions was reflected in community composition and diversity, in which the bloom was different and less diverse from the residual brine population. The distributional patterns of microbial genes suggested Dead Sea community trends in mono- and divalent cation metabolisms as well as in transposable elements. This may indicate possible mechanisms and pathways enabling these microbes to survive in such a harsh environment.

Giardia duodenalis is an intestinal parasite of many vertebrates. The presence of G. duodenalis in the marine environment due to anthropogenic and wildlife activity is well documented, including the contributions from untreated sewage and storm water, agricultural run-off and droppings from terrestrial animals. Recently, studies have detected this protistan parasite in the faeces of marine vertebrates such as whales, dolphins, seals and shore birds. To explore the population biology of G. duodenalis in marine life, we determined the prevalence of G. duodenalis in two species of seal (Halichoerus grypus, Phoca vitulina vitulina and Phoca vitulina richardsi) from the east and west coasts of the USA, sequenced two loci from G. duodenalis-positive samples to assess molecular diversity and examined G. duodenalis distribution amongst these seals and other marine vertebrates along the east coast. We found a significant difference in the presence of G. duodenalis between east and west coast seal species. Only the zoonotic lineages of G. duodenalis, Assemblages A and B and a novel lineage, which we designated as Assemblage H, were identified in marine vertebrates. Assemblages A and B are broadly distributed geographically and show a lack of host specificity. Only grey seal (Halichoerus grypus) samples and one gull sample (Larus argentatus) from a northern location of Cape Cod, Massachusetts, USA, showed the presence of Assemblage H haplotypes; only one other study of harbour seals from the Puget Sound region of Washington, USA previously recorded the presence of an Assemblage H haplotype. Assemblage H sequences form a monophyletic clade that appears as divergent from the other seven Assemblages of G. duodenalis as these assemblages are from each other. The discovery of a previously uncharacterised lineage of G. duodenalis suggests that this parasite has more genetic diversity and perhaps a larger host range than previously believed.

PCR-based surveys of microbial communities commonly use regions of the small-subunit ribosomal RNA (SSU rRNA) gene to determine taxonomic membership and estimate total diversity. Here we show that the length of the target amplicon has a significant effect on assessments of microbial richness and community membership. Using operational taxonomic unit (OTU)- and taxonomy-based tools, we compared the V6 hypervariable region of the bacterial SSU rRNA gene of three amplicon libraries of c. 100, 400 and 1000 base pairs (bp) from each of two hydrothermal vent fluid samples. We found that the smallest amplicon libraries contained more unique sequences, higher diversity estimates and a different community structure than the other two libraries from each sample. We hypothesize that a combination of polymerase dissociation, cloning bias and mispriming due to secondary structure accounts for the differences. While this relationship is not linear, it is clear that the smallest amplicon libraries contained more different types of sequences, and accordingly, more diverse members of the community. Because divergent and lower abundant taxa can be more readily detected with smaller amplicons, they may provide better assessments of total community diversity and taxonomic membership than longer amplicons in molecular studies of microbial communities.

Actin genic regions were isolated and characterized from the heterokont-flagellated protists, Achlya bisexualis (Oomycota) and Costaria costata (Chromophyta). Restriction enzyme and cloning experiments suggested that the genes are present in a single copy and sequence determinations revealed the existence of two introns in the C. costata actin genic region. Phylogenetic analyses of actin genic regions using distance matrix and maximum parsimony methods confirmed the close evolutionary relationship of A. bisexualis and C. costata suggested by ribosomal DNA (rDNA) sequence comparisons and reproductive cell ultrastructure. The higher fungi, green plants, and animals were seen as monophyletic groups; however, a precise order of branching for these assemblages could not be determined. Phylogentic frameworks inferred from comparisons of rRNAs were used to assess rates of evolution in actin genic regions of diverse eukaryotes. Actin genic regions had nonuniform rates of nucleotide substitution in different lineages. Comparison of rates of actin and rDNA sequence divergence indicated that actin genic regions evolve 2.0 and 5.3 times faster in higher fungi and flowering plants, respectively, than their rDNA sequences. Conversely, animal actins evolve at approximately one-fifth the rate of their rDNA sequences.

Small subunit rRNA sequences have been determined for 10 of the most clinically important pathogenic species of the yeast genus Candida (including Torulopsis [Candida] glabrata and Yarrowia [Candida] lipolytica) and for Hansenula polymorpha. Phylogenetic analyses of these sequences and those of Saccharomyces cerevisiae, Kluyveromyces marxianus var. lactis, and Aspergillus fumigatus indicate that Candida albicans, C. tropicalis, C. parapsilosis, and C. viswanathii form a subgroup within the genus. The remaining significant pathogen, T. glabrata, falls into a second, distinct subgroup and is specifically related to S. cerevisiae and more distantly related to C. kefyr (psuedotropicalis) and K. marxianus var. lactis. The 18S rRNA sequence of Y. lipolytica has evolved rapidly in relation to the other Candida sequences examined and appears to be only distantly related to them. As anticipated, species of several other genera appear to bear specific relationships to members of the genus Candida.

The complete small subunit rRNA gene sequences of the heterotrich Blepharisma americanum and the colpodid Colpoda inflata were determined to be 1719 and 1786 nucleotides respectively. The phylogeny produced by comparisons with other ciliates indicated that C. inflata is allied more closely with the nassophoreans and oligohymenophoreans than the spirotrichs. This is consistent with the placement of the colpodids in the Class Copodea. Blepharisma americanum was not grouped with the hypotrichs but instead was placed as the earliest branching ciliate. The distinct separation of B. americanum supports the elevation to class status given the heterotrichs based on morphological characters.

Oceanic lithosphere exposed at the sea floor undergoes seawater– rock alteration reactions involving the oxidation and hydration of glassy basalt. Basalt alteration reactions are theoretically capable ofsupplyingsufficientenergyforchemolithoautotrophicgrowth 1 . Such reactions have been shown to generate microbial biomass in the laboratory 2 , but field-based support for the existence of microbes that are supported by basalt alteration is lacking. Here, using quantitative polymerase chain reaction, in situ hybridization and microscopy, we demonstrate that prokaryotic cell abundances on seafloor-exposed basalts are 3–4 orders of magnitude greater than in overlying deep sea water. Phylogenetic analyses of basaltic lavas from the East Pacific Rise (96 N) and around Hawaii reveal that the basalt-hosted biosphere harbours high bacterial community richness and that community membership is shared between these sites. We hypothesize that alteration reactions fuel chemolithoautotrophic microorganisms, which constitute a trophic base of the basalt habitat, with important implications for deep-sea carbon cycling andchemical exchange between basalt and sea water. We assessed the abundance, species richness and phylogenetic

We applied nucleic acid-based molecular methods, combined with estimates of biomass (ATP), pigments, and microelectrode measurements of chemical gradients, to map microbial diversity vertically on a millimeter scale in a hypersaline microbial mat from Guerrero Negro, Baja California Sur, Mexico. To identify the constituents of the mat, small-subunit rRNA genes were amplified by PCR from community genomic DNA extracted from layers, cloned, and sequenced. Bacteria dominated the mat and displayed unexpected and unprecedented diversity. The majority (1,336) of the 1,586 bacterial 16S rRNA sequences generated were unique, representing 752 species (≥97% rRNA sequence identity) in 42 of the main bacterial phyla, including 15 novel candidate phyla. The diversity of the mat samples differentiated according to the chemical milieu defined by concentrations of O 2 and H 2 S. Bacteria of the phylum Chloroflexi formed the majority of the biomass by percentage of bulk rRNA and of clones in rRNA gene libraries. This result contradicts the general belief that cyanobacteria dominate these communities. Although cyanobacteria constituted a large fraction of the biomass in the upper few millimeters (>80% of the total rRNA and photosynthetic pigments), Chloroflexi sequences were conspicuous throughout the mat. Filamentous Chloroflexi bacteria were identified by fluorescence in situ hybridization within the polysaccharide sheaths of the prominent cyanobacterium Microcoleus chthonoplastes , in addition to free living in the mat. The biological complexity of the mat far exceeds that observed in other polysaccharide-rich microbial ecosystems, such as the human and mouse distal guts, and suggests that positive feedbacks exist between chemical complexity and biological diversity.

Studies of deeply buried, sedimentary microbial communities and associated biogeochemical processes during Ocean Drilling Program Leg 201 showed elevated prokaryotic cell numbers in sediment layers where methane is consumed anaerobically at the expense of sulfate. Here, we show that extractable archaeal rRNA, selecting only for active community members in these ecosystems, is dominated by sequences of uncultivated Archaea affiliated with the Marine Benthic Group B and the Miscellaneous Crenarchaeotal Group, whereas known methanotrophic Archaea are not detectable. Carbon flow reconstructions based on stable isotopic compositions of whole archaeal cells, intact archaeal membrane lipids, and other sedimentary carbon pools indicate that these Archaea assimilate sedimentary organic compounds other than methane even though methanotrophy accounts for a major fraction of carbon cycled in these ecosystems. Oxidation of methane by members of Marine Benthic Group B and the Miscellaneous Crenarchaeotal Group without assimilation of methane–carbon provides a plausible explanation. Maintenance energies of these subsurface communities appear to be orders of magnitude lower than minimum values known from laboratory observations, and ecosystem-level carbon budgets suggest that community turnover times are on the order of 100–2,000 years. Our study provides clues about the metabolic functionality of two cosmopolitan groups of uncultured Archaea.

Sulfite reductases are key enzymes of assimilatory and dissimilatory sulfur metabolism, which occur in diverse bacterial and archaeal lineages. They share a highly conserved domain "C-X5-C-n-C-X3-C" for binding siroheme and iron-sulfur clusters that facilitate electron transfer to the substrate. For each sulfite reductase cluster, the siroheme-binding domain is positioned slightly differently at the N-terminus of dsrA and dsrB, while in the assimilatory proteins the siroheme domain is located at the C-terminus. Our sequence and phylogenetic analysis of the siroheme-binding domain shows that sulfite reductase sequences diverged from a common ancestor into four separate clusters (aSir, alSir, dsr, and asrC) that are biochemically distinct; each serves a different assimilatory or dissimilatory role in sulfur metabolism. The phylogenetic distribution and functional grouping in sulfite reductase clusters (dsrA and dsrB vs. aSiR, asrC, and alSir) suggest that their functional diversification during evolution may have preceded the bacterial/archaeal divergence.

Small-subunit (SSU) rRNA gene sequences were obtained by PCR from 12 Blastocystis isolates from humans, rats, and reptiles for which elongation factor 1α (EF-1α) gene sequences are already available. These new sequences were analyzed by the Bayesian method in a broad phylogeny including, for the first time, all Blastocystis sequences available in the databases. Phylogenetic trees identified seven well-resolved groups plus several discrete lineages that could represent newly defined clades. Comparative analysis of SSU rRNA- and EF-1α-based trees obtained by maximum-likelihood methods from a restricted sampling (13 isolates) revealed overall agreement between the two phylogenies. In spite of their morphological similarity, sequence divergence among Blastocystis isolates reflected considerable genetic diversity that could be correlated with the existence of potentially ≥12 different species within the genus. Based on this analysis and previous PCR-based genotype classification data, six of these major groups might consist of Blastocystis isolates from both humans and other animal hosts, confirming the low host specificity of Blastocystis . Our results also strongly suggest the existence of numerous zoonotic isolates with frequent animal-to-human and human-to-animal transmissions and of a large potential reservoir in animals for infections in humans.

Two new species of heterolobosean amoebae from anoxic environments, Monopylocystis visvesvarai and Sawyeria marylandensis, are described on the basis of light microscopy, electron microscopy, and their phylogenetic affiliation based on analyses of nuclear small-subunit ribosomal RNA gene sequences. Both species lack mitochondria but have organelles provisionally interpreted as hydrogenosomes, and neither can tolerate aerobic conditions. As their conditions of culture do not exclude all oxygen, they may be microaerophiles rather than strict anaerobes. Both species have unusual nucleolar morphologies. Monopylocystis visvesvarai, from a marine sediment, has nucleolar material distributed around the nuclear periphery. It is the first non-aerobic heterolobosean protist for which a cyst is known; the cyst is unmineralized and unornamented except for a single, raised, plugged pore. Sawyeria marylandensis, from an iron-rich freshwater stream, has nucleolar material distributed in one or two parietal masses, which persist during mitosis. In phylogenetic analyses of small-subunit rRNA gene sequences, Monopylocystis visvesvarai, Sawyeria marylandensis and Psalteriomonas lanterna converge to form a single clade of non-aerobic (anaerobic/microaerophilic) heteroloboseans.

Giardia lamblia is an early branching eukaryote, and although distinctly eukaryotic in its cell and molecular biology, transcription and translation in G. lamblia demonstrate important differences from these processes in higher eukaryotes. The cyclic octapeptide amanitin is a relatively selective inhibitor of eukaryotic RNA polymerase II (RNAP II) and is commonly used to study RNAP II transcription. Therefore, we measured the sensitivity of G. lamblia RNAP II transcription to alpha-amanitin and found that unlike most other eukaryotes, RNAP II transcription in Giardia is resistant to 1 mg/ml amanitin. In contrast, 50 microg/ml amanitin inhibits 85% of RNAP III transcription activity using leucyl-tRNA as a template. To better understand transcription in G. lamblia, we identified 10 of the 12 known eukaryotic rpb subunits, including all 10 subunits that are required for viability in Saccharomyces cerevisiae. The amanitin motif (amanitin binding site) of Rpb1 from G. lamblia has amino acid substitutions at six highly conserved sites that have been associated with amanitin resistance in other organisms. These observations of amanitin resistance of Giardia RNA polymerase II support previous proposals of the mechanism of amanitin resistance in other organisms and provide a molecular framework for the development of novel drugs with selective activity against G. lamblia.

The Guaymas Basin (Gulf of California) is a hydrothermal vent site where thermal alteration of deposited planktonic and terrestrial organic matter forms petroliferous material which supports diverse sulfate-reducing bacteria. We explored the phylogenetic and functional diversity of the sulfate-reducing bacteria by characterizing PCR-amplified dissimilatory sulfite reductase ( dsrAB ) and 16S rRNA genes from the upper 4 cm of the Guaymas sediment. The dsrAB sequences revealed that there was a major clade closely related to the acetate-oxidizing delta-proteobacterial genus Desulfobacter and a clade of novel, deeply branching dsr sequences related to environmental dsr sequences from marine sediments in Aarhus Bay and Kysing Fjord (Denmark). Other dsr clones were affiliated with gram-positive thermophilic sulfate reducers (genus Desulfotomaculum ) and the delta-proteobacterial species Desulforhabdus amnigena and Thermodesulforhabdus norvegica . Phylogenetic analysis of 16S rRNAs from the same environmental samples resulted in identification of four clones affiliated with Desulfobacterium niacini , a member of the acetate-oxidizing, nutritionally versatile genus Desulfobacterium , and one clone related to Desulfobacula toluolica and Desulfotignum balticum . Other bacterial 16S rRNA bacterial phylotypes were represented by non-sulfate reducers and uncultured lineages with unknown physiology, like OP9, OP8, as well as a group with no clear affiliation. In summary, analyses of both 16S rRNA and dsrAB clone libraries resulted in identification of members of the Desulfobacteriales in the Guaymas sediments. In addition, the dsrAB sequencing approach revealed a novel group of sulfate-reducing prokaryotes that could not be identified by 16S rRNA sequencing.

Complete 16S-like ribosomal RNA coding regions were obtained from the fungal symbiont of five genera of attine (leaf-cutting) ants and two free-living fungi. Phylogenetic analyses with distance matrix, maximum likelihood, and parsimony methods revealed that the attine fungal symbionts are homobasidiomycetes in the order Agaricales. Comparison of the topology of the attine fungal symbiont phylogenetic tree with a tree based on attine ant morphology revealed a congruent branching pattern of the more derived attine ants and their fungal symbionts. The parallel branching pattern suggests a long-term coevolution of derived leaf-cutting attine ants and their fungal symbionts.

Group II introns are catalytic RNAs and mobile retrotransposable elements known to be present in the genomes of some nonmarine bacteria and eukaryotic organelles. Here we report the discovery of group II introns in a bacterial mat sample collected from a deep-sea hydrothermal vent near 9°N on the East Pacific Rise. One of the introns was shown to self-splice in vitro. This is the first example of marine bacterial introns from molecular population structure studies of microorganisms that live in the proximity of hydrothermal vents. These types of mobile genetic elements may prove useful in improving our understanding of bacterial genome evolution and may serve as valuable markers in comparative studies of bacterial communities.

Diplomonads, such as Giardia, and their close relatives retortamonads have been proposed as early-branching eukaryotes that diverged before the acquisition-retention of mitochondria, and they have become key organisms in attempts to understand the evolution of eukaryotic cells. In this phylogenetic study we focus on a series of eukaryotes suggested to be relatives of diplomonads on morphological grounds, the "excavate taxa". Phylogenies of small subunit ribosomal RNA (SSU rRNA) genes, alpha-tubulin, beta-tubulin, and combined alpha- + beta-tubulin all scatter the various excavate taxa across the diversity of eukaryotes. But all phylogenies place the excavate taxon Carpediemonas as the closest relative of diplomonads (and, where data are available, retortamonads). This novel relationship is recovered across phylogenetic methods and across various taxon-deletion experiments. Statistical support is strongest under maximum-likelihood (ML) (when among-site rate variation is modeled) and when the most divergent diplomonad sequences are excluded, suggesting a true relationship rather than an artifact of long-branch attraction. When all diplomonads are excluded, our ML SSU rRNA tree actually places retortamonads and Carpediemonas away from the base of the eukaryotes. The branches separating excavate taxa are mostly not well supported (especially in analyses of SSU rRNA data). Statistical tests of the SSU rRNA data, including an "expected likelihood weights" approach, do not reject trees where excavate taxa are constrained to be a clade (with or without parabasalids and Euglenozoa). Although diplomonads and retortamonads lack any mitochondria-like organelle, Carpediemonas contains double membrane-bounded structures physically resembling hydrogenosomes. The phylogenetic position of Carpediemonas suggests that it will be valuable in interpreting the evolutionary significance of many molecular and cellular peculiarities of diplomonads.

Molecular microbial ecology studies have revealed remarkable prokaryotic diversity in extreme hydrothermal marine environments. There are no comparable reports of culture-independent surveys of eukaryotic life in warm, anoxic marine sediments. By using sequence comparisons of PCR-amplified small subunit ribosomal RNAs, we characterized eukaryotic diversity in hydrothermal vent environments of Guaymas Basin in the Gulf of California. Many sequences from these anoxic sediments and the overlaying seawater represent previously uncharacterized protists, including early branching eukaryotic lineages or extended diversity within described taxa. At least two mechanisms, with overlapping consequences, account for the eukaryotic community structure of this environment. The adaptation to anoxic environments is evidenced by specific affinity of environmental sequences to aerotolerant anaerobic species in molecular trees. This pattern is superimposed against a background of widely distributed aerophilic and aerotolerant protists, some of which may migrate into and survive in the sediment whereas others (e.g., phototrophs) are simply deposited by sedimentary processes. In contrast, bacterial populations in these sediments are primarily characteristic of anoxic, reduced, hydrocarbon-rich sedimentary habitats.

This paper presents the first molecular phylogenetic analysis of the phylum Ctenophora, by use of 18S ribosomal RNA sequences from most of the major taxa. The ctenophores form a distinct monophyletic group that, based on this gene phylogeny, is most closely related to the cnidarians. Our results suggest that the ancestral ctenophore was tentaculate and cydippid-like and that the presently recognized order Cydippida forms a polyphyletic group. The other ctenophore orders that we studied (Lobata, Beroida, and Platyctenida) are secondarily derived from cydippid-like ancestors, a conclusion that is also supported by developmental and morphological data. The very short evolutionary distances between characterized ctenophore 18S rRNA gene sequences suggests that extant ctenophores are derived from a recent common ancestor. This has important consequences for future studies and for an understanding of the evolution of the metazoans.

A phylogenetic analysis of protein disulfide isomerase (PDI) domain evolution was performed with the inclusion of recently reported PDIs from the amitochondriate protist Giardia lamblia, yeast PDIs that contain a single thioredoxin-like domain, and PDIs from a diverse selection of protists. We additionally report and include two new giardial PDIs, each with a single thioredoxin-like domain. Inclusion of protist PDIs in our analyses revealed that the evolutionary history of the endoplasmic reticulum may not be simple. Phylogenetic analyses support common ancestry of all eukaryotic PDIs from a thioredoxin ancestor and independent duplications of thioredoxin-like domains within PDIs throughout eukaryote evolution. This was particularly evident for Acanthamoeba PDI, Dictyostelium PDI, and mammalian erp5 domains. In contrast, gene duplication, instead of domain duplication, produces PDI diversity in G. lamblia. Based on our results and the known diversity of PDIs, we present a new hypothesis that the five single-domain PDIs of G. lamblia may reflect an ancestral mechanism of protein folding in the eukaryotic endoplasmic reticulum. The PDI complement of G. lamblia and yeast suggests that a combination of PDIs may be used as a redox chain analogous to that known for bacterial Dsb proteins.